Nucleotide deduced amino acid sequence, isolation and purification of heat-shock chlamydial proteins

ABSTRACT

The present invention relates to novel polypeptides comprising a unique “chlamydial-specific” primary structural conformation and one or more of the biological properties of eukaryotic or prokaryotic stress-response proteins which are characterized by being the expressed products of an endogenous or exogenous DNA sequence in a eukaryotic or prokaryotic host cell. Sequences coding for part or all of the amino acid residues of the chlamydial HypA or HypB protein or for analogs thereof may be incorporated into autonomously replicating vectors employed to transform or transfect suitable procaryotic or eukaryotic host cells such as bacteria or vertebrate cells in culture. The HypB protein is a member of the family of stress response proteins referred to as HSP60. Products of expression of the DNA sequences display the identical physical, immunological, and histological properties as the chlamydial proteins isolated from natural, non-recombinant, organisms.

This is a division of application Ser. No. 07/841,323, filed Feb. 25,1992, abandoned, which is a division of application Ser. No. 07/679,302filed Apr. 2, 1991, abandoned, which is a division of application Ser.No. 07/531,317, filed May 31, 1990, now U.S. Pat. No. 5,071,962.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention pertains in general to stress-response proteinsand to polynucleotides encoding such factors. The present applicationpertains in particular to the HypB protein of chlamydia bacteria,specifically to the HypB protein from Chlamydia psittaci and Chlamydiatrachomatis, to fragments and polypeptide analogs thereof, and topolynucleotides encoding the same. The HypB protein is a member of ahighly conserved family of stress response proteins referred to asHSP60. The HypB protein has a number of acronyms such as the chlamydialGroEL, the chlamydial 57kD antigen and the chlamydial HSP60.

2. Background Information

Members of the genus Chlamydia are obligate intracellular bacteria thatare differentiated from all other prokaryotes by their uniqueintracellular growth cycle. Two species of Chlamydia exist; C.trachomatis, strictly a human pathogen, and C. psittaci, a pathogen oflower mammals. Chlamydiae primarily infect mucosal epithelia, and inhumans C. trachmatis causes a formidable group of infections, some ofwhich can progress to severe complications including blindness,infertility, and perhaps arthritis. The most significant of these in thenumbers of people afflicted is trachoma, the leading cause ofpreventable blindness in the world (Jones, Trans. Ophthalmol.Soc.(U.K.), 95, 16(1975)).

Though the pathogenic events that lead to development of severe andoften debilitating, post-infection sequelae are not known, animmunological mechanism has been suggested based on studies of humantrachoma and sub-human primate models of ocular chlamydial infection(Dawson, Human Chlamydial Infections (1978); Grayston, et al., Rev.Infect. Dis., 7,717 (1985); Silverstein, Invest. Ophthalmol., 13, 560(1974); Collier, Arch. ges. Virusforsch., 22, 280 (1967)).

Early studies in humane and in sub-human primates indicate that priorvaccination with killed chlamydiae frequently results in more severetrachoma upon reinfection (Wang, et al., Am. J. Ophthalmol., 63, 1615(1967); See Wang, et al., Am. J. Ophthalmol., 63, 1133 (1967); Grayston,et al., Ann. N.Y. Acad. Sci., 98, 352 (1962); Woolridge, et al., Am. J.Ophthalmol., 63, 1645 (1967); Bell, et al., Am. J. Trop. Med. Hyg. 18,568 (1969)). Moreover, in some individuals with trachoma, chlamydialantigens and DNA are detected in conjunctival tissue in the absence ofcultivatable chlamydia (Wilson, et al., Arch. Ophthalmol., 104, 688(1986); Schachter, et al., J. Infect. Dis., 158 1347 (1989)). These datasupport the hypothesis of an immunologically mediated pathogenesis.

C. trachomatis infection of non-human primates and C. psittaci infectionof guinea pigs are good model systems for studying chlamydialpathogenesis. Previous studies using these models show that repeatedexposure to infectious chlamydiae is necessary to establish the chronicinflammation characteristic of trachoma (Monnickendam, et al., Br. J.Ophthalmol., 64, 284 (1980); Taylor, et al., Invest. Ophthalmol. Vis.Sci., 23, 507 (1982)). Repeated challenge with infectious chlamydiaeresults in an atypical infection of shortened duration in whichchlamydia are difficult to reisolate, and severe ocular disease results;thus suggesting that immune responses are partly protective, but alsodeleterious. Repeated infection produces a submucosal cellularinfiltrate of lymphocytes and macrophages (Patton, et al., J. Infect.Dis., 153, 870 (1986), Monnickendam, supra, and Taylor, supra) like thatobserved in individuals with trachoma (Hogan &. Zimmerman, OphthalmicPathology, 240-244 (1962)). Collectively, the human and animal studiesargue for a pathogenic role of delayed hypersensitivity (DH)′ inchlamydial disease.

The most direct evidence for DH in pathogenesis of chlamydial diseasecomes from the observations that a crude extract of viable chlamydiaeelicits severe ocular inflammation in immune animals (Watkins, et al.,Proc. Natl. Acad. Sci. (USA), 83, 7480 (1986); Taylor, et al., J.Immunol., 138, 3023 (1987)).

In immune guinea pigs, this extract produces an ocular inflammatoryresponse whose histopathology is consistent with human trachoma andchlamydial-induced tubal infertility. Those results support thehypothesis that the host's immune response to chlamydial infection is,in part, deleterious (Moller, et al., Br. J. Vener. Dis. 55, 422 (1979);Hogan, supra, and Watkins, supra).

The inflammation elicited by a chlamydial antigen was clinically andhistologically identical to that caused by primary infection whichsuggested the pathogenesis of the ocular disease was immunologicallymediated. This also suggested that the pathogenesis of recurrentchlamydial disease was not due to active infection (Watkins, supra).

Identifying hypersensitivity as a major pathogenetic mechanism wasimportant not only in understanding chlamydial associated diseaseprocesses but also in establishing future strategies to controlchlamydial diseases by immunoprophylaxis. Ocular delayedhypersensitivity has been shown to be induced at mucosal surfaces otherthan conjunctival-i.e., intestinal or vaginal. Primary chlamydialinfection at one mucosal site can elicit a delayed hypersensitivityreaction at either the same or different mucosal surfaces and cancontribute to the pathogenesis of chlamydial disease in humans (Watkins,supra).

Recent data suggested that the pathogenesis of guinea pig inclusionconjunctions (GPIC) was mediated by delayed hypersensitivity to anantigen common to strains of both chlamydial species (Watkins, supra).

Prior to the present invention, the biologically active antigen had notbeen identified. As indicated above, a crude chlamydial extract elicitedocular and dermal delayed hypersensitivity. Lipopolysaccharide (LPS) isa major component of this crude extract and was a suspected antigen.Purified LPS did not elicit hypersensitivity in immune animals but thisdiscovery did not preclude the possibility that the allergen wascomposed of a complex of LPS with protein, carbohydrate, or lipid(Watkins, supra). Development of a vaccine for trachoma and otherchlamydial diseases, which would preclude the deleterious immuneresponse, required identification of the antigen.

The major outer membrane protein (MOMP) is the major structural proteinof chlamydiae and is immunogenic (Taylor, et al. J. Immunol., 138, 3023(1987), supra). Purified MOMP, however, did not elicit an ocular DHresponse in immune animals. The crude extract of chlamydial elementarybodies (EBs), as indicated above, was biologically active but the exactnature of the extract remained to be determined since neither LPS norMOMP (components of this extract) elicited an inflammatory response inpurified form. Prior to the present invention, the antigenic determinantand inflammatory response elicited by the prude extract was thought tobe caused by LPS in conjunction with an alternation in membranepermeability induced by the extraction solution (Taylor, supra). Thestimulus for the inflammation response in trachoma has been the subjectof ongoing speculation.

The Applicants have isolated and purified a chlamydial antigenic proteinresponsible for the ocular delayed hypersensitivity inflammatoryresponse from Chlamydia psittaci and Chlamydia trachomatis. Theimmunologically bioactive component is the HypB protein of the presentinvention (Morrison, et al., J. Exp. Med., 169, 663 (1989)). Alsodescribed is a HypA antigenic protein (Morrison, et al., supra). Thechlamydial gene of the C. psittaci and C. trachomatis that encodes theHypB protein have been cloned, and the recombinantly produced protein ofC. psittaci has been shown to elicit an ocular DH response in immuneguinea pigs. The sequencing of the gene revealed a close relatedness tothe heat-shock or stress proteins GroEL of Escherichia coli, HtpB ofCoxiella burnetii,. 65 k of Mycobacterium tuberculosis, and Hsp60 ofSaccharomyces cerevisiae.

While several antigenic detection diagnostic tests for chlamydia areavailable, none use immunological reagents specific for the HypB proteinof the present invention (Chernesky, et al., J. Infect. Dis. 154, 141(1986); Howard, et al., J. Clin. Microbiol., 23, 329 (1986); Tam, etal., N. Engl. J. Med., 310, 1146 (1984)). The present invention allowsfor the preparation of peptides analogous to chlamydial-specificdeterminants for use in monoclonal antibody and monospecific-polyclonalantisera preparation which can be used to identify chlamydiae inclinical specimens or cell culture of chlamydial isolates. Syntheticpeptides analogous to the chlamydial-specific determinants can be usedin serological assays to diagnose chlamydial infections. The HypBchlamydial protein elicits a cell-mediated immune response in additionto an antibody response. Therefore, the present invention allows for theuse of a chlamydial specific region of the protein as a skin testantigen to diagnose or monitor chlamdial infection; analogous to the useof the TB skin test antigen PPD.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a segment of a DNAmolecule which codes for a HypB and/or a HypA protein derived from thebacteria Chlamydia and the encoded proteins.

It is another object of the present invention to provide a recombinantDNA molecule comprising a vector and a segment of a DNA molecule whichcodes for a HypB protein derived from the bacteria Chlamydia.

It is a further object of the present invention to provide a host cellstably transformed or transfected with the DNA vector provided by thisinvention in a manner allowing expression of the protein encoded by thevector.

It is yet another object of the present invention to provide a method ofproducing the HypB chlamydial antigen on a commercial scale.

It is another object of the present invention to provide a method forthe detection of chlamydial HypB protein in clinical specimens, cellcultures or mammals and a method for the diagnosis of chlamydialinfection in individuals.

Various other objects and advantages of the present invention willbecome obvious from the drawings and the detailed description of theinvention.

In one embodiment, the present invention relates to a DNA segmentencoding all, or a unique portion, of a HypB Chlamydia protein.

In another embodiment, the present invention relates to a DNA segmentencoding all, or a unique portion, of a HypA Chlamydia protein.

In another embodiment, the present invention relates to a substantiallypure form of a chlamydial HypB protein eliciting a delayed ocular anddermal inflammatory response in mammals.

In a further embodiment, the present invention relates to arecombinantly produced protein having all, or a unique portion, of theamino acid sequence given in FIG. 5 or FIG. 7.

In yet another embodiment, the present invention relates to antibodiesspecific for the chlamydial HypB protein.

In a further embodiment, the present invention relates to a recombinantDNA molecule comprising a DNA segment encoding all, or a unique portion,of a HypB Chlamydia protein and a vector.

In yet a further embodiment, the present invention relates to a hostcell stably transformed with the above recombinant DNA molecule in amanner allowing expression of the protein encoded in the recombinant DNAmolecule.

In another embodiment, the present invention relates to a method ofproducing a Chlamydia HypB protein comprising culturing host cellsstably transformed with a recombinant DNA molecule comprising a DNAsegment encoding all, or a unique portion, of the HypB Chlamydia proteinand vector, in a manner allowing expression of the DNA segment andthereby production of the protein.

In a further embodiment, the present invention relates to a method forthe detection of the chlamydial HypB protein in a sample which methodcomprises contacting a reagent which specifically reacts with thechlamydial HypB protein with the sample under conditions such thatreaction between the chlamydial protein and the agent can occur, anddetecting the presence or absence of a reaction.

In yet a further embodiment, the present invention relates to a vaccinefor mammals against chlamydial disease comprising all, or a uniqueportion of a Chlamydia HypB protein in an amount sufficient to induceimmunization against the disease, and a pharmaceutically acceptablecarrier.

The entire contents of all publications mentioned herein are herebyincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Restriction endonuclease map of the 7.2-kb chlamydial DNA insertof pGP57. The internal 2.0-kb EcoRI fragment (E1) is indicated. Openboxes represent the hypA and hypB ORFS. The direction of transcriptionis indicated by an arrow.

FIG. 2. SDS-PAGE and immunoblot analysis of E. coli strain JM109[pUC12]and recombinant strain JM109[pGP57]. (A) Coomassie brilliantblue-stained gel of whole-cell lysates. (B) Immunoblot probed withmonospecific polyclonal anti-57-kD serum. Recombinant polypeptides areindicated by approximate M, values (57 k and 20 k). Immunologicalreagents specific for the 20-kD polypeptide were not available, and itwas not reactive with hyperimmune anti-GPIC serum.

FIG. 3. In vitro transcription-translation analysis of purified plasmidDNAs and immunoprecipitation of in vitro translated polypeptide. Onemicrogram of purified plasmid DNA was used as suggested by themanufacturer or the commercial in vitro translation kit (AmerishamCorp.). Reaction proceeded at 37° C. for 45 min, followed by a 5 minchase with nonradiolabelled methionine. Reactions mixtures weresubjected to SDS-PAGE and analyzed directly by fluorography (lanes 1, 2,and 3), or immunoprecitated with polyclonal monospecific anti-57kD serum(lanes 4, 5, and 6) or normal rabbit serum (lanes 7, 8, and 9), thenanalyzed. Lanes 1, 4 and 7, no DNA added to reaction mixture: lanes 2, 5and 8, pUC12 DNA; and lanes 3, 6 and 9, pGP57 DNA. *indicates 57-kD(HypB) and 20-kD (HypA) recombinant polypeptides.

FIG. 4. Ocular DH response elicited by chlamydial antigen preparations.Ocular hypersensitivity was assessed at 24 h after antigen challenge andscored as described in Example 2. TX100 buffer, TX-100 GPIC and nativeHypB protein were prepared as described in Morrison et al. The nativeand recombinant HypB proteins were mixed 1:1 with 2X TX-100 buffer andtested for hypersensitivity. Inflammatory index is the mean responsefrom eight guinea pigs per group. Naive animals were challenged withantigen preparation, and in all instances, the inflammatory index was<1.

FIG. 5. Nucleotide sequence of the C. psittaci strain GPIC hyp operon.The deduced amino acid sequences of the hypA and hypB ORFs are indicatedabove the nucleotide sequence. The inferred promoter region (−35 regionand −10 region), ribosomal binding sites (single underscore), and thedyad symmetry (arrows) of the proposed transcription terminator areindicated. The sequence corresponding to the oligonucleotides used forNorthern blot analysis are indicated (====). Numbers to the right of thefigure refer to nucleotide position, and amino acid numbering is abovethe deduced amino acid sequence.

FIG. 6. Comparison of the deduced amino acid sequences of (A) the C.psittaci HypA protein and the C. burnetii HtpA and E. coli GroESproteins, and (B) the C. psittaci HypB protein and the C. burnetti HtpB,E. coli GroEL, M. tuberculosis 65 k and S. cerevisiae Hsp 60 proteins.Identity is represented by a period (.), conserved amino acid changes bya colon (:) above the amino acid, and gaps introduced for alignmentpurposes are represented by a hyphen (-). *, the Hsp60 sequence isaligned beginning at amino acid number 22. Amino acid position numberdoes not directly correspond with the amino acid numbers in FIG. 5because of the inserted gaps needed for alignment.

FIG. 7. Nucleotide and deduced amino acid sequence of the C. trachomatisserovar A hyp operon.

FIG. 8. Immunoblot analysis of whole-cell lysates probed withmonospecific anti-57-kD serum. Each sample consisted of 20 μg of totalprotein. Lane 1, C. psittaci strain GPIC; lane 2, C. trachomatis serovarB; lane 3, C. trachomatis serovar L2; lane. 4, E. coli strain JM109;lane 5, E. coli strain JM109[pGp57]; lane 6, S. typhimurium strainSL3261; lane 7, N. gonorrhoeae strain MS11;lane 8, E. rickettsii Rstrain; lane 9, C. burnetii strain Nine Mile; lane 10, B. burgdorferistrain B31; and lane 11, M. tuberculosis strain H37RA.

FIG. 9. Comparison of the the deduced amino acid sequence of the C.trachomatis serovar A HypA protein and the C. psittaci strain GPIC HypAprotein. Amino acid identity is indicated by a period (.) and conservedchanges by a colon (:).

FIG. 10. Comparison of the deduced amino acid sequence of the C.trachomatis serovar A HypB protein and the C. psittaci strain GPIC HypBprotein. Amino acid identity is indicated as in FIG. 9.

FIG. 11. Chlamydial specificity of the polyclonal rabbit anti-57-kDantiserum and the anti-45-kD mAb. (A) Coomassie brilliant blue-stainedgel of all 15 serovars of C. trachomatis and two strains of C. psittaci,Mn, and GPIC. The MOMPs of each vary in M, and are the major stainingpolypeptides indicated by the bracket. (B) Immunoblot probed withpolyclonal monospecific anti-57-kD antiserum. (C) Immunoblot probed withmAb GPIC IV-B1. The polyclonal anti-57-kD and mAb anti-45-kD antibodieswere monospecific and reacted with proteins found on all 15 C.trachomatis serovars and two C. psittaci strains. In some serovars the45-kD protein comigrated with the MOMP (serovars A, C, H, I, and J) andwas thus difficult to distinguish on the Coomassie-stained gel. However,it could be distinguished when probed with the anti-45-kD antibody. The57-and 45-kD proteins are major genus-specific proteins found on EBs.

FIG. 12. Immunoblot analysis of immunoaffinity purification of the HypBand 45-kD genus-specific chlamydial proteins. 10 ml of the solubleTX-100 extract of GPIC EBs (10¹⁰ EBs) were sequentially passed throughthe anti-45 kD column followed by passage through the anti-57-kD column.The columns were washed and adherent proteins were eluted as describedin Example 6. The 45-kD and HypB proteins were eluted as homogeneousprotein preparations as determined by immunoblotting and Coomassiebrilliant blue and silver staining of SDS-PAGE gels (data not shown).Immunoblot probed with A, polyclonal anti-GPIC EB antiserum; B, anti-45kD mAb; and C, polyclonal monospecific anti-57-kD antiserum. Lane 1,GPIC EBs; lane 2, soluble Triton X-100 extract of GPIC EBs; lane 3,TX-100 extract after passage through the anti-45-kD affinity column;lane 4, antigens eluted from the anti-45-kD column; lane 5, TX-100extract after passage through the anti-45-kD and anti-57-kD column; lane6, antigens eluted from the anti-57-kD affinity column.

FIG. 13. Reactivity of monoclonal anti-HypB antibodies (mAbs). The genusspecificity of the mabs was shown using Western blot assay. All mAbsreacted with both the native chlamiydial HypB protein; lanes 1 and 5,and the recombinant HypB protein lanes 2 and 4. However, the mAbs didnot react with the homologous E. coli protein (lane 3).

FIG. 14. Species specificity of the moncolonal antibodies isdemonstrated using Western blot assay.

FIG. 15. Hematoxylin and eosin-stained sections of the palpebralconjunctiva from ocular immune guinea pigs 24 h after challenged with(A) TX-100 buffer; (B) soluble TX-100 extract of GPIC EBs; (C)immunoaffinity-purified HypB protein; and (D) TX-100 extract of GPIC EBsdepleted of the HypB and 45-kD proteins. See footnotes to Table I(Example 7) for dosages of antigen administered. M, mucosal epithelium;SM, submucosa; P, polymorphonuclear neutrophil; L, lymphocyte, Mφ,macrophage.

DETAILED DESCRIPTION

The present invention relates to segments of DNA which encode a HypBprotein and a HypA protein derived from the bacteria Chlamydia, and tounique portions (i.e., at least 15 nucleotides) of such segments. TheHypB proteins of C. psittaci and C. trachomatis have a molecular weight,as determined by sequence analysis, of about 58 kilo Daltons (kD). TheseHypB proteins have the same characteristics, function and are about 95%homologous. The HypA proteins of the two species have a molecular weightas determined by sequence analysis of about 12 kD.

The invention also relates the HypB chlamydial bacterial protein encodedin the hyp B gene, which is characterized as follows: (a) it isendogenous to and extractable from chlamydial elementary bodies (EBs)and reticulate bodies (RBs), (b) it produces delayed ocular and dermalinflammatory responses in mammals, (c) it is reactive with antiserumproduced by antigens extracted from chlamydial EBs, (d) it can bepurified essentially free of other material by standard biochemical andimmunological techniques, (e) it has a molecular weight in SDS PAGE gelsof about 57,000 D, (f) it is highly soluble, (h) it is very immunogenic,(i) it contains chlamydial specific epitopes, and (j) it containsepitopes common to other ca. 60 kD stress-response proteins. In oneembodiment, the protein of the invention has the amino acid sequence setforth in FIG. 5. In another embodiment, the HypB protein of the presentinvention has the amino acid sequence as set forth in FIG. 7. Indeed,the chlamydial HypA and HypB proteins are homologues of the E. coliGroES and GroEL heat shock proteins (HSP), respectively; and the HypBprotein is a member of a widely conserved family of prokaryotic andeukaryotic stress response proteins referred to as ESP60. The inventionalso relates to unique portions of the HypB chlamydial bacterial proteindescribed above, for example, a unique portion (at least 5 or 6 aminoacids) of the sequence set forth in FIG. 5 or FIG. 7. The HypB proteinsubstantially free of proteins with which it is normally associated canbe bound to a solid support such as, for example, agarose, sepharose,plastic, nylon membrane or nitrocellulose paper.

The HypA protein encoded in the hyp A gene has a molecular weight in SDSPAGE gels of about 18,000 to 20,000 D. In one embodiment, it has theamino acid sequence as set out in FIG. 5, or a unique portion thereof.In another embodiment, it has the amino acid sequence as set forth inFIG. 7, or a unique portion thereof.

The present invention also relates to a recombinant DNA moleculecomprising a vector and the above-described DNA segment which encodes atleast the HypB protein or unique portion thereof. Possible vectorsinclude plasmids, for example pUC8, and other vectors known in the arthost cells stably transformed or transfected with the above-describedrecombinant DNA molecule in a manner which allows expression of the hypproteins, fragment or analog. Examples of appropriate host cells includeprokaryotic and eukaryotic cells depending on the vector used.

In another embodiment, the present invention relates to a process forthe production of a polypeptide product having part or all of theprimary structural conformation and/or biological activity of thenaturally occurring hyp chlamydial proteins comprising growing hostcells transformed or transfected with the above-described recombinantDNA molecule (for example pGP57) in a manner which allows the expressionof the hyp gene and thereby, production of the polypeptide product, andisolating the desired polypeptide product.

In another embodiment, the present invention relates to antibodies(monoclonal and polyclonal) specific for the HypB chlamydial antigen.Monospecific polyclonal antisera of the invention reacts with eukaryoticor prokaryotic antigens with molecular weights of approximately 57 kDand with proteins analogous to the chlamydial-specific determinant. Theinvention further relates to antibodies specifically for HypA chlamydialprotein. The antibodies of the invention can be prepared using methodsknown in the art. The antibodies can be bound to a solid support suchas, for example, agarose, sepharose, plastic nylon membranes ornitrocellulose paper. Antibodies can be prepared using, for example,native HypB, recombinant HypB, unique recombinant portions of HypB andsynthetic peptides.

The present invention also relates to the detection of Chlamydia in asample. The sample can be a clinical specimen, such as a swab of aninfected site, or a cell culture. The presence of a chlamydiaL HypA orHypB protein is detected by contacting a reagent which specificallyreacts with the protein with the sample under conditions such that areaction can be effected and detected. Possible reagents includeantibodies specific for the protein.

The presence of a DNA or RNA sequence encoding a chlamydia HypA or HypBprotein is detected by contacting a labelled DNA probe which hybridizesto the sequence with the sample under conditions such that hybridizationoccurs and is detected.

The present invention also relates to a vaccine for use in mammalsagainst chlamydial disease. In one embodiment of this aspect of thisinvention, as is customary for vaccines, the HypB protein of the presentinvention can be delivered to a mammal in a pharmacologically acceptablevehicle. As one skilled in the art will understand, it is not necessaryto use the entire protein. A unique portion of the protein (for example,a recombinantly produced polypeptide corresponding to a portion of theHypB protein) can be used. Vaccines of the present invention can includeeffective amounts of immunological adjuvants known to enhance an immuneresponse. The protein or polypeptide is present in the vaccine in anamount sufficient to induce an immune response against the antigenicprotein and thus to protect against chlamydia infection. Protectiveantibodies are usually best elicited by a series of 2-3 doses givenabout 2 to 3 weeks apart. The series can be repeated when circulatingantibodies concentration in the patient drops.

The invention is described in further detail in the followingnon-limiting Examples.

EXAMPLE 1 Construction of Genomic Library, Selection, Subcloning andSequencing.

C. psittaci (GPIC) genomic DNA isolated from 6×10¹⁰ IFU, was partiallydigested with Sau3A, and sized by electrophoresis on a 0.7% agarose gel(Nano, et al., Infect. Immun., 48, 372 (1985)). 5 to 10-kilobase (kb)fragments were electroeluted and ligated into BamHI-digested, alkalinephosphate-treated pUC8 (Boehringer Mannheim Biochemicals, Indianapolis,Ind.) (Vieira, et al., Gene, 19, 259 (1982)). E. coli strain JM109 wastransformed with the recombinant plasmids, grown in Luria brothsupplemented with 250 μg/ml of carbenicillin, and screened by colonyblot using hyperimmune anti-GPIC rabbit serum (Hanahan, J. Mol. Biol.,166, 557 (1983). Helfman, et al., Proc. Natl. Acad. Sci. (USA), 80, 31(1983)). An immunoreactive clone, JM109 [pGP57], was isolated andanalyzed by SDS-PAGE and immunoblotting. Two highly expressedrecombinant products, 57-kD and 20-kD polypeptides, were visualized inCoomassie blue-stained gels of whole-cell lysates (FIG. 2). The 7.2-kbGPIC insert of pGP57 was restriction mapped (FIG. 1) and shown tohybridize with GPIC DNA by Southern blot analysis. An internal 2.0-kbEcoRI fragment (E1) was subcloned into pTZ18R and shown to produce animmunoreactive polypeptide of 50 kD, presumably a truncated version ofthe 57-kD protein found in pGP57. A partial sequence of the 2.0-kbchlamydial DNA fragment from the E1 subclone was obtained by thedideoxy-chain termination method using pUC forward and reverse universalprimer following the manufacturer's suggested procedures (Sequenase,United States Biochemical Corp., Cleveland, Ohio). After obtaining apartial DNA sequence from the E1 subclone, sequencing was continuedusing synthetic oligonucleotide primers (SAM1, Milligen-Biosearch, Inc.,San Rafael, Calif.), and purified pGP57 plasmid DNA.

The cloning and sequencing methods described above were also performedusing C. trachomatis serovar A genomic DNA. In the C. trachomatisexperiments the plasmid pUC18 was used as the cloning vector. E. colistrain JM 109 was tranfected with the recombinant pUC18 plasmids. Animmunoreactive clone, JM109[pTA571], was isolated and analyzed in thesame manner as clone JM109[pGP57].

Sequence Analysis

A 2.4-kb GPIC DNA insert of pGP57 carries two open reading frames (ORF)whose deduced amino acid sequences are presented in FIG. 5. Sequencesconsistent with Shine-Dalgarno ribosomal binding sites (AGGA) precededthe ATG initiation codons of both ORFs. One ORF spanned 306 nucleotidesand encoded a polypeptide of 102 amino acids [relative molecular mass(M_(r)) 11,202], and the other spanned 1,632 nucleotides to encode apolypeptide of 544 amino acids (M_(r) 58,088).

The serovar A DNA insert of pTA571 also carries two ORF whose deducedamino acid sequences are shown in FIG. 7. One ORF has a relativemolecular mass of 17,000 daltons and the other ORF has a relativemolecular mass of 57,000 daltons on denaturing SDS-polyacrylamide gels.

Because the 57-kD protein has a single known function, its ability toelicit an immunopathological response in primed animals (a DH response),the whole operon has been termed hyp (for hypersensitivity); C. psittacihypA encodes the 11.2-kD protein and hypB encodes the 58.1-kD protein.The apparent molecular mass of HypA and HypB proteins on denaturingpolyacrylamide gels is 20 kD and 57 kD, respectively. The presumptiveTAA translational terminator sequence of hypA was followed by anintergenic region of 50 bases. The larger ORF, hypB, terminated at a TAAstop codon followed by sequences resembling a rho-independent terminator(Platt, Cell, 24, 10 (1981)).

The nucleotide sequence of the hyp operon from C. trachomatis serovar Ais shown in FIG. 7. The operon contains two open reading frames (hyp Aand hyp B), which encode polypeptides of calculated molecular weights ofapproximately 11.1 kD (HypA) and 58 kD (HypB). The deduced amino acidsequences of the C. trachomatis serovar A HypA and HypB polypeptides areshown in FIGS. 7, 9 and 10.

At nucleotide position −231 of the C., psittaci hyp operon sequence likea heat shock promoter (−35 region, T-C-C-CTTGAA, −10 region, CCCCAT-T-)was found (Christman, et al., Cell, 41, 753 (1985)). There wasconsiderable sequence agreement for the −10 region, with only a single Gfor C substitution. The 3′ end of the −35 region was in completeagreement, but the 5′ half was not conserved. No other upstreamconsensus promoter regions were found. Although this inferred promoterregion has similarities with promoters of genes for other heat-shockproteins, a temperature dependent expression of the polypeptides encodedby this recombinant operon in E. coli has not been demonstrated.Expression of the two proteins in bacteria grown at 22° C. is high, andmay result from the high copy number of pGP57.

Because of the tandem hypA and hypB ORFs and their strikingresemblance-to the E. coli. groE and the C. burnetti htp operons,Northern hybridizations were done to determine whether both hypA andhypB sequences were contained in a single transcript. Oligonucleotideprobes complementary to the 5′ end of hypA, and the 3′ end of hypB (FIG.5) revealed that hypA and hypB are expressed as a single mRNA transcriptof ≈2300 nucleotides.

Predicted Amino Acid Sequence Homology

The amino acid sequence encoded by the C. psittaci HypA protein showedidentify with HtpA (42%) and GroES (38%) proteins (FIG. 6A) (Hemmingsen,et al., Nature, 333, 330 (1988); Vodkin et al., J. Bacteriol., 170, 1227(1988)). The C. psittaci HypB protein showed more identity to the HtpBprotein of C. burnetii (61%), the GroEL protein of E. coli (60%), the65-kD protein of M. tuberculosis (58%), and the mature Hsp 60 protein ofS. cerevisiae (53%) (Vodkin, supra. Hemmingsen, supra. Shinnick, J.Bacteriol., 169, 1080 (1987). Reading, et al., Nature, 337, 655 (1989)).Regions of identity were scattered throughout the sequence. However, theN- and C- terminal sequences, sequence 318 to 361, and sequence 421 to481 exhibited more divergence and may be determinants of the polypeptidethat specify chlamydial-specific epitopes. The predicted amino acidsequence of the C. trachomatis HypA and HypB proteins were 85 and 94%identical with the C. psittaci HypA and HypB proteins, respectively(FIG. 9 and 10).

Because the 57-kD chlamydial protein showed considerable amino acididentity with the common GroEL antigen of E. coli., other prokaryoticorganisms were examined by immunoblotting with anti-57-kD serum (FIG.8). This antiserum reacted with polypeptides of similar M_(r)(M_(r)=molecular weight determined in SDS PAGE gels) in all bacteriaexamined. These results along with the amino acid homologies demonstratethat the 57-kD chlamydial protein is a member of the family of widelyconserved stress-response proteins referred to as common antigen (Hoiby,Scand. J. Immunol., 4(2), 187 (1975)).

EXAMPLE 2 Antigen Preparation, Purification and Ocular DH

10⁹ chlamydial elementary bodies were washed three times with saline,resuspended in 10 ml of PBS containing 0.5% Triton X-100 (TX-100),incubated at 37° C. for 30 min and sonicated for 3 to 5 min. (Watkinssupra). Insoluble material was removed by centrifugation at 100,000×gand the HypB protein was purified from the soluble extract byimmunoaffinity chromatography (Morrison, supra). Briefly, the solubleextract of chlamydial elementary bodies was passed over an affinitycolumn prepared with monospecific anti-57-kD rabbit serum. Morrison,supra. The column was washed with 10 volumes of PBS containing 0.5%Triton X-100 and 0.5 M NaCl. Absorbed antigen was eluted with 3.0 Mpotassium thiocyanate, dialyzed against PBS, and analyzed by SDS-PAGEand immunoblotting. A single 57-kD polypeptide was seen by Coomassieblue staining, and it reacted with monospecific anti-57kD serum byimmunoblot analysis.

The ability of these antigen preparations to elicit an ocular DHresponse was assessed by placing 25 μl of antigen preparation ( 2 to 6μg of protein) onto the lower conjunctival sac of ocular immune guineapigs (Morrison, supra). The hypersensitivity response was assessedclinically at 24 h, and scored using a scale of 0 to 4: 0, negative; 1,slight hyperemia and edema of the lower palpebral conjunctivae; 2,hyperemia and edema of the lower palpebral conjunctiva with slighthyperemia of the bulbar conjunctivae; 3, overt hyperemia and edema ofthe lower palpebral and bulbar conjunctivae; 4, same as 3 with theaddition of mucopurulent exudate (Morrison, supra).

Ocular Delayed Hypersensitivity Elicited by the Recombinant HypB Protein

Immune guinea pigs, previously infected with GPIC and recovered, werechallenged with a soluble extract of JM109, JM109(pGP57) or theimmunoaffinity purified recombinant HypB antigen (purified as describedfor elementary bodies i.e. above in example 2). Both the soluble extractof JM109[pGP57] and the purified recombinant HypB protein elicited anocular DH response when administered topically to the conjunctivae ofimmune but not naive guinea pigs (FIG. 4). Severity of inflammationresembled that elicited by a crude extract of C. psittaci EBs andimmunoaffinity purified native 57-kD protein.

EXAMPLE 3 Production of the Recombinant Polypeptide Products

Organisms.

The C. trachomatis serovars B/TW-5 and L2/LGV-434, and C. psittacistrain guinea pig inclusion conjunctivitis (GPIC) were grown in HeLa 229cells, and EBs were purified by discontinuous density centrifugation inRenografin (E. R. Squibb and Sons, Princeton, N.J.) (Caldwell, et al.,Infect. Immun. 31, 1161 (1981)). Inclusion-forming units (IFU) weredetermined by methods described in Sabet, et al. (J. Clin. Microbiol.,20, 217 (1984)). E. coli strain JM109, and pUC8 and pTZ18R plasmids havebeen described previously by Yanisch-Perron, et al. (Gene, 33,103(1985), Vieira at 259, supra, and Pharmacia LKB Biotechnology, Inc.,Piscataway, N.J.).

Identification and Characterization of Recombinant Clones

Recombinant colonies were screened by blotting with a polyclonalantiserum to GPIC. One recombinant, JM109[pGP57], expressed two productswith apparent molecular weights of 57 kD and 20 kD (FIG. 2A); the 57-kDspecies reacted with a polyclonal monospecific anti-57-kD serum byimmunoblotting (FIG. 2B). An E. coli polypeptide of the same size wasalso recognized by this antiserum. To verify that the 20- and 57-kDpolypeptides were encoded by the plasmid and were not due to anincreased expression of the E. coli proteins, in vitrotranscription-translation was done. The recombinant plasmid, pGP57,encoded two polypeptides of 57 kD and 20 kD (FIG. 3), and the in vitrosynthesized 57-kD polypeptide immunoprecipitated with anti-57-kD serum.A recombinant clone expressing polypeptides similar to those of pGP57has been briefly described elsewhere (Menozzi, et al., FEMS Microbiol.Lett., 58, 59 (1989)).

EXAMPLE 4 SDS-PAGE, Electrophoretic Transfer, and Immunoblotting

SDS-PAGE was performed using 12.5% polyacrylamide gels as described byDreyfuss et al., except chlamydial whole-cell lysates and samples wereprepared with 2X Laemmli sample buffer (Dreyfuss, et al., Mol. Cell.Biol., 4,415 (1984), Laemmli, Nature, 227,680 (1970)). The apparentmolecular masses of chlamydial proteins recognized by the anti-57-kD andanti-45-kD antibodies were determined by comparing the migrationdistances of these immunoreactive proteins with a plot of migrationdistance vs. the log molecular mass of several protein standards(Bio-Rad Laboratories, Richmond, Calif.). Electrophoretic transfer andprocessing were done as described by Zhang et al. J. Immunol., 138, 575(1987)).

In Vitro Detection of Recombinant Polypeptides

In vitro transcription-translation of pGP57 was performed according tothe manufacturers instructions (Amersham Corp., Arlington Heights,Ill.). ³⁵S-methionine labelled products were analyzed by SDS-PAGE andfluorography as described in Bonner, et al. (Eur. J. Biochem., 46, 83(1974) and Zhang, supra). The in vitro transcription-translationmixtures were also subjected to immunoprecipitation with a monospecificanti-57-kD rabbit serum or normal rabbit serum, and analyzed by SDS-PAGEand fluorography (Morrison, supra. Bonner, et al., Eur. J. Biochem., 46,83 (1974) and Zhang, supra).

Characteristics of Chlamydial Proteins

The SDS-PAGE polypeptide profile of the 15 serovars of C. trachomatisand two strains of C. psittaci are shown in FIG. 11A. The HypB and 45-kDproteins are indicated by arrows and the MOMPs are the major stainingprotein bands that appear in the bracketed area of the gel. The genusspecificity of the anti-57-kD and anti-45-kD antibodies are demonstratedin FIG. 11B and C, respectively. The anti-57-kD antiserum reacted with asimilar molecular mass protein in all chlamydial strains tested.Similarly, the anti-45-kD antibody reacted with all strains, althoughmore strongly with the 45-kD protein of the C. psittaci strains GPIC andMn. The weaker reactivity of the C. trachomatis strains might beexplained by quantitative differences in this protein among strains ormay simply reflect stronger reactivity to the immunizing species. Slightvariability in M_(r) of this protein among strains was also observed.These data demonstrate the genus specificity of the anti-57-kD andanti-45-kD antibodies, and the prominence of these proteins inchlamydial EBs.

EXAMPLE 5 Affinity Chromatography and Antigen Purification

The protein A binding fraction of a polyclonal monospecific rabbitantiserum against the genus-specific 57-kD chlamydial protein and a mAb(purified IgG), reactive against the 45-kD genus-specific chlamydialprotein (GPIC-IV B1, IgG, a gift from Dr. You-Xun Zhang, Rocky MountainLaboratories), were used to prepare the affinity columns. The polyclonalanti-57-kD antiserum was prepared by immunizing rabbits with isolatedimmunoprecipitin bands excised from two-dimensionalimmunoelectropherograms as described in Caldwell, et al. (J. Immunol.115, 969 (1975)).

The immunopreciptates used as immunogen in the preparation of theanti-57-kD antiserum correspond to the single common crossreactingantigen observed by crossed immunoelectrophoresis (Caldwell at 963,supra). mAb GPIC IV-B1 was prepared by immunizing BALB/c mice with GPICEBs and following the procedures described in Caldwell, et al. (Infect.Immun., 44, 306 (1984)).

The purified antibodies were covalently crosslinked to the supportmatrix (Schrieder, et al., J. Biol. Chem., 257, 10766 (1982)). 1-mlpacked volume of swollen protein A-Sepharose CL-4B (Sigma Chemical Co.,St. Louis, Mo.) beads was gently mixed with 10 mg (at 1 mg/ml in 50 mMPBS, pH 7.2) of either anti-45-kD or anti-57-kD antibody at 22° C. for45 min. The immunomatrix (protein A-Sepharose antibody) was washed threetimes with 100 mM borate buffer, pH 8.2, followed by a single 20-ml washwith 200 mM triethanolamine, pH 8.2. The antibody was covalentlycrosslinked to the protein A-Sepharose by resuspending the immunomatrixin 20 ml of freshly prepared 20 mM dimethylpimelimidate-dihydrochloridein 200 mM triethanolamine, pH 8.2, and gently mixed for 45 min at 22° C.The immunomatrix was pelleted by light centrifugation and resuspended in1.0 ml of 20 mM ethanolamine, pH 8.2. After 5 min at 22° C., theimmunomatrix was washed once with 10 ml of 100 mM borate buffer, pH 8.2,poured into a column, washed with 20 ml of PBS, and stored at 4° C.until used. The 45 and 57-kD chlamydial proteins were purified from aTriton X-100 soluble extract of GPIC EBs. Morrison, supra.

10 ml of the soluble GPIC extract was preabsorbed with 0.1 g of proteinA-Sepharose for 45 min at 22° C. to remove nonspecifically bindingcomponents of the extract. The preabsorbed antigen extract wassequentially passed through the anti45-kD and anti-57-kD columns,respectively. Each column was washed with 20 ml of 50 mM phosphatebuffer, pH 7.2, containing 500 mM NaCl and 0.5% Triton X-100. Boundantigen was eluted with 3.0 M potassium thiocyanate (KSCN) in PBS. 1-mlfractions were collected, dialyzed overnight against PBS at 4° C., andanalyzed for purity by SDS-PAGE and immunoblotting. Approximately 500and 300 μg of protein were eluted from the anti-45-kD and anti-57-kDcolumns, respectively. Fractions containing purified protein wereassayed for their ability to elicit ocular hypersensitivity as describedabove.

Immunoblot Analysis of Purified Chlamydial Antigens

The 45-kD and HypB chlamydial proteins and LPS are genus-specificconstituents and major components found in the soluble fraction of theTX-100 extract of GPIC EBs. This extract causes an ocularhypersensitivity response in ocular immune guinea pigs (Watkins, supra).Since a major genus-specific constituent of this extract (LPS) failed toinduce ocular hypersensitivity, the extract was purified usingimmunoaffinity chromatography to obtain the 45-kD and HypB proteins(FIG. 12). The soluble TX-100 extract of GPIC EBs contains a number ofimmunoreactive proteins recognized by antiserum raised to GPIC EBs (FIG.12, lane 2). Passage of this extract through the anti-45-kD columnfollowed by passage through the anti-57-kD column efficiently removedthe 45-kD and HypB proteins (FIG. 12, lanes 3 and 5, respectively). Theywere then eluted from the columns as antigenically homogeneous proteins(FIG. 12, lanes 4 and 6). Homogeneity of the protein preparations wasalso demonstrated by Coomassie brilliant blue and silver staining ofSDS-PAGE gels. Noteworthy is the finding that both the 45-kD and HypBproteins migrate as single bands in EB preparations, but were observedas doublets in the extract and purified fractions.

EXAMPLE 6

Chlamydial Infection, and Ocular Hypersensitivity

Male and female Hartley guinea pigs, 8-12 wk old from a chlamydial-freecolony, were used throughout these studies. Animals were bred andmaintained at the Rocky Mountain Laboratories, Hamilton, Mont. Animalswere infected by placing 10 μl containing 10 ID₅₀ (10×10² IFU) of GPIConto the lower conjunctiva as described in Watkins, supra.

Conjunctivae of infected guinea pigs were culture negative by 4 wk afterinfection. These guinea pigs are referred to as ocular immune and wereused to test for ocular hypersensitivity 6-8 wk after primary infection.Ocular hypersensitivity was assessed by placing 25 μl of the appropriateantigen solution onto the lower conjunctival sac. The hypersensitivityresponse was assessed clinically at 2, 12, 18, 24, 48, and 72 h and wasscored using a scale of 0 to 4 (Watkins, supra, Morrison, supra; pg. 14,supra). Peak inflammation was observed at 24 h after instillation ofantigen. The time course of the inflammatory response and the nature ofthe cellular infiltrate (see FIG. 13) has led us to refer to thisresponse as an ocular DH.

Ocular Hypersensitivity Elicited by Chlamydial Antigen Preparations

Chlamydial antigen preparations and affinity-purified proteins weretested for their ability to elicit an ocular inflammatory response inimmune and naive guinea pigs (see Morrison et al., J of Exp Med 169,663-675 (1989)). The purified HypB, but not the 45-kD chlamydialprotein, elicited an inflammatory response when administered topicallyto the conjunctival surface of ocular immune guinea pigs. The intensityof the inflammatory response elicited by the purified HypB protein (3.1)was similar to that elicited by the soluble TX-100 extract (3.4).Depleting the extract of the 45-kD and HypB proteins did not render theextract noninflammatory. However, the intensity of the ocularinflammation was marginal (2.3) and waned more quickly than the responseelicited by the extract containing these proteins.

EXAMPLE 7 Monoclonal antibodies to the HypB protein

The HypB protein was isolated from the recombinant clone (JM109[pGP57])by SDS-polyacrylamide gel electrophoresis and electroelution, and usedto immunize BALB/C mice. Hybridomas secreting anti-57kD antibodies wereproduced by fusing splenic lymphocytes from immunized mice with murinemyeloma cells (P3-NS-1AG-4/1) using standard procedures (Caldwell, etal. Infec. Immun. 44: 306 (1984)). Eight monoclonal antibodies wereisolated, all of which are of the IgG₁, isotype. The reactivity of themAbs using Western blot assay is shown in FIGS. 13 and 14. FIG. 13demonstrates the genus specificity of the mABs. All mABs reacted withthe chlamydial protein, that is, both the native HypB protein foundassociated with chlamydial organisms, lanes 1 and 5, and the recombinantHypB proteins, lanes 2 and 4, but failed to react with the homologousprotein found in E. coli. (lane 3).

FIG. 14 illustrates the species specificity of the mABs. mABs GP57-5,GP57-16, GP57-19 and GP57-23 react equally well with all C. trachomatisserovars and the two C. psittaci strains and mABs GP57-12 and GP57-21vary in reactivity among C. trachomatis and C. psittaci strains.

EXAMPLE 8

The C. trachomatis serovars A/Har-13, B/TW-5, Ba/Apa-2, C/TW-3, D/UW-31,E/Bour, F/IC-Cal-13, G/UW-57, H/UW-4, I/UW-12, J/UW-36, K/UW-31,L1/LGV-440, L2/LGV-434, and L3/LGV-404, C. psittaci strains guinea piginclusion conjunctivitis (GPIC), and meningopneumonitis (Mn) were grownin HeLa 229 cells, and EBs were purified by discontinuous densitycentrifugation in Renografin (E. R. Squibb and Sons, Princeton, N.J.)(Caldwell, et al., Infect. Immun., 31, 1161 (1981)). inclusion-formingunits (IFU) were determined by methods described previously). (Sabet,supra).

Histology

Guinea pigs were killed with T-61 euthanasia solution (Hoechst Corp.,Somerville, N.J.). The upper and lower eyelids were removed, fixed inneutral-buffered 10% formalin, and stained with hematoxylin and eosin asdescribed in Watkins, supra.

Histological Profile of Ocular Hypersensitivity Responses

To determine the cellular characteristics of the inflammation elicitedby the various antigen preparations, hematoxylin- and eosin-stainedsections of the palpebral conjunctiva were examined at the time of peakinflammation (24 h post-challenge) (FIG. 15). The inflammatory responseelicited by the soluble TX-100 extract and the purified genus-specificHypB protein were indistinguishable (FIG. 15, B and C). Both thesepreparations elicited a subacute inflammatory response characterized bylymphoid hyperplasia and a submucosal infiltrate consisting primarily ofmononuclear macrophages and lymphocytes. Occasional polymorphonuclearneutrophils (PMN) were observed at the mucosal surface.

In contrast, the inflammatory response elicited by the extract depletedof the 45-kD and HypB proteins was more acute and characterized by amarked PMN infiltrate (FIG. 15D).

In some instances, DH responses in the guinea pig have been shown to theexamples of cutaneous basophil hypersensitivity. In fact, certainantigens elicit conjunctival cutaneous basophil hypersensitivity(Allansmith, et al., J. Allergy Clin. Immunol., 78, 919 (1986)).Therefore, Giemsa-stained sections of the palpebral conjunctiva fromchlamydial-antigen-challenged guinea pigs were examined for the presenceof basophils. Only very few basophils (1%) were observed in theinfiltrates. Thus, because the inflammatory response elicited by theTX-100 extract and the purified HypB protein was primarily mononuclear(macrophage and lymphocyte) and delayed in appearance (24 h), we havecharacterized it as an ocular DH.

Hybridomas producing monoclonal antibodies to the HypB protein weredeposited on Apr. 4, 1990 under the Budapest Treaty at the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852.mAb19-2E1 referred to as mAB GP-57-19, hereinabove and mAb5-2G9 referredto as mAB GP57-5, hereinabove were assigned the accession number ATCCHB10407 and ATCC HB10408 respectively.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention.

What is claimed is:
 1. An isolated DNA molecule encoding a HypBChlamydia protein, wherein said molecule consists of a nucleotidesequence set forth as nucleotide 360 to nucleotide 1991 of the sequenceshown in FIG. 5 or consists of a nucleotide sequence set forth asnucleotide 347 to nucleotide 1978 of the sequence shown in FIG.
 7. 2. Avector comprising the DNA molecule according to claim
 1. 3. The vectoraccording to claim 2 wherein the vector is selected from the groupconsisting of a plasmid, a bacteriophage and a eucaryotic virus vector.4. A host cell stably transformed or transfected with the vectoraccording to claim 2 in a manner allowing expression of the HypBchlamydia protein.
 5. The host cell according to claim 4 wherein saidhost cell is a procaryotic cell or a eucaryotic cell.
 6. The host cellaccording to claim 5 wherein said procaryotic cell is an Escherichiacoli cell.
 7. A method of producing a Chlamydia HypB protein comprising,culturing host cells according to claim 4, in a manner allowingexpression of said DNA segment and thereby production of said proteinfrom said host cells.
 8. The vector of claim 2, wherein the DNA segmentencoding the HypB Chlamydia protein consists of the sequence set forthas nucleotide 360 to nucleotide 1991 of the sequence shown in FIG.
 5. 9.The vector of claim 2, wherein the DNA segment encoding the HypBChlamydia protein consists of the sequence set forth as nucleotide 347to nucleotide 1978 of the sequence shown in FIG.
 7. 10. An isolated DNAmolecule encoding a HypA Chlamydia protein, wherein said moleculeconsists of a nucleotide sequence set forth as nucleotide 1 tonucleotide 306 of the sequence shown in FIG. 5 or consists of anucleotide sequence set forth as nucleotide 1 to nucleotide 306 of thesequence shown in FIG.
 7. 11. A vector for the introduction of DNA intoeucaryotic or procaryotic host cells comprising: the DNA moleculeaccording to claim
 10. 12. The vector of claim 11, wherein said vectoris a plasmid, bacteriophage or eucaryotic virus vector.
 13. A host cellstably transformed or transfected with the vector of claim 11 in amanner allowing expression of the DNA molecule.
 14. The host cellaccording to claim 13, wherein said host cell is a procaryotic cell or aeucaryotic cell.
 15. The host cell according to claim 14, wherein saidhost cell is an Escherichia coli cell.
 16. A recombinant nucleic acidmolecule comprising: a nucleic acid sequence encoding a HypA Chlamydiaprotein consisting of an amino acid sequence as shown in FIG. 9; or (b)a nucleic acid sequence encoding a HypB Chlamydia protein consisting ofan amino acid sequence as shown in FIG.
 10. 17. The recombinant nucleicacid molecule of claim 16, wherein the nucleic acid sequence encodes theHypA Chlamydia protein consisting of an amino acid sequence as shown inFIG.
 9. 18. The recombinant nucleic acid molecule of claim 16, whereinthe nucleic acid sequence encodes the HypB Chlamydia protein consistingof an amino acid sequence as shown in FIG.
 10. 19. A vector comprisingthe recombinant nucleic acid molecule of claim
 16. 20. The vectoraccording to claim 19, wherein the vector is selected from the groupconsisting of a plasmid, a bacteriophage and a eucaryotic virus vector.21. A host cell stably transformed or transfected with the vectoraccording to claim
 19. 22. The host cell according to claim 21 whereinsaid host cell is a procaryotic cell or a eucaryotic cell.
 23. The hostcell according to claim 22 wherein said procaryotic cell is anEscherichia coli cell.
 24. A method of producing a Chlamydia proteincomprising, culturing host cells according to claim 21, in a mannerallowing expression of said nucleic acid sequence and thereby productionof said Chlamydia protein from said host cells.